Surface-Independent, Surface-Modifying, Multifunctional Coatings and Applications Thereof

ABSTRACT

The present invention provides a surface-independent surface-modifying multifunctional biocoating and methods of application thereof. The method comprises contacting at least a portion of a substrate with an alkaline solution comprising a surface-modifying agent (SMA) such as dopamine so as to modify the substrate surface to include at least one reactive moiety. In another version of the invention, a secondary reactive moiety is applied to the SMA-treated substrate to yield a surface-modified substrate having a specific functionality.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/939,386filed Jul. 11, 2013, which is a divisional of U.S. application Ser. No.11/875,237 filed Oct. 19, 2007, and has issued as U.S. Pat. No.8,541,060 on Sep. 24, 2013, which claims priority to U.S. ProvisionalApplication 60/853,013, filed Oct. 19, 2006, the entirety of all ofwhich are incorporated by reference herein for all purposes.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant DE 014193awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF INVENTION

This invention generally relates to polymeric coatings. Moreparticularly, this invention relates to surface-independent,surface-modifying, multifunctional coatings.

BACKGROUND OF THE INVENTION Surface Modification

Chemical modification of bulk material substrates plays a central rolein modern chemical, biological and material sciences, as well as inapplied sciences, engineering and technology. Methods for chemicalmodification of bulk material substrates have developed by interfacialchemistry using organothiol-metals, enediol-oxides, silane-oxides, andother physicochemical methods, in which the predominant purpose is toimpose desired properties on non-functional substrates. Moleculesutilized for surface modification mostly have bifunctional end groups inwhich one end anchors to substrates and the other end provides chemicalfunctionality to the substrate surface.

The existing toolbox for functional modification of material/substratesurfaces includes methods such as self-assembled monolayer (SAM)formation, functionalized silanes, Langmuir-Blodgett deposition,layer-by-layer assembly, and genetically-engineered surface-bindingpeptides. Although widely implemented in research, these conventionalmethods have limitations for widespread practical use. For instance,chemical specificity between interfacial modifiers and substrates (e.g.,alkanethiols on noble metals and silanes on oxides) is typicallyrequired, complex instrumentation is typically required, and thesubstrate size/shape (Langmuir-Blodgett deposition) is often limited, ormulti-step procedures for implementation (layer-by-layer assembly andsurface-binding genetically engineered peptides) are required. Moreimportantly, the substrates available for conventional surfacemodification chemistry is the primary limitation.

Mussel Adhesive

Mussels represent a natural surface-independent adhesive. Mussels arepromiscuous fouling organisms which attach to virtually all types ofinorganic and organic substrates, including classicallyadhesion-resistant materials such as polytetrafluoroethylene (PTFE)(FIG. 1A). Mussels' adhesive versatility may lie in the amino acidcomposition of proteins found near the plaque-substrate interface (FIG.1B-D), which is rich in 3,4-dihydroxy-L-phenylalanine (DOPA) and lysineamino acids. DOPA participates in reactions leading to bulksolidification of the adhesive and forms strong covalent andnon-covalent interactions with substrates.

Dopamine is a small molecule compound that contains both catechol (DOPA)and amine (lysine) groups (FIG. 1E). Dopamine can be electro-polymerizedonto conducting substrates (Y. Li, et al., Thin Solid Films, 497, 270,2006).

Needed in the art of surface modification is a method ofsurface-independent modification of a substrate whereby specificfunctional moieties can be displayed on the surface.

SUMMARY OF THE INVENTION

In the present invention, it is shown that dopamine and relatedcompounds can act as a powerful building block for thin polymer filmdeposition on virtually any bulk material surface wherein the depositedfilms are easily adaptable for a remarkable variety of functional uses.In one embodiment the deposition is spontaneous.

In one preferred embodiment, the present invention is a novelsurface-independent, surface-modification method whereby substrates aremodified to display at least one reactive moiety on the substratesurface by contacting at least a portion of the substrate with asurface-modifying agent (SMA). Because of the surface-independent natureof the present method, specific applications include diverse fields suchas biocompatible coatings of medical devices, surface modifications ofdrug delivery carriers and tissue engineering scaffolds, biosensors,industrial and consumer coatings, semiconductors, surface catalysts andnext generation electronic displays.

In a first embodiment, the present invention pertains to a method ofmodifying a substrate surface, the method comprising contacting at leasta portion of the substrate with an alkaline solution under oxidativeconditions, the solution comprising a surface-modifying agent (SMA)according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10; wherein y ranges from 0 to 10, provided that x or yis at least 1; and wherein the substrate surface is modified. In apreferred embodiment, the SMA forms a polymeric coat on the substratesurface.

The SMA may also be selected from the group consisting of3,4-dihydroxy-L-phenylalanine (DOPA), 3,4-dihydroxyphenylalanine methylester, dopamine, norepinephrine and epinephrine, and may be an aqueoussolution.

In one embodiment of the SMA, x and y are both 1 and R₁ and R₄ form adouble bond when eliminated. However, in alternative embodiments of theSMA, one of R₁ or R₄ is a halide, a hydroxyl or a thiol and one of R₃ orR₅ is a hydrogen atom, and R₂ is NH₂ or NHR, wherein R is an alkyl oraromatic group. In further alternate embodiments of the SMA, x is 1, yis 1, R₁ is a hydroxyl, R₃, R₄ and R₅ are hydrogen. In still furtheralternate embodiments of the SMA, x and y are each 1, each of R₁, R₃, R₄and R₅ are hydrogen atoms, and R₂ is an NH₂ or NHR, where R is an alkylor aromatic group; or, alternatively, one of R₁ or R₄ is a halide, ahydroxyl or a thiol and one of R₃ or R₅ is a hydrogen atom.

In alternate embodiments of the SMA, x+y is at least 2, x+y is at least3, and x+y ranges from 1 to 6.

In alternate embodiments of the SMA, hydroxyls of the phenyl moiety arepositioned at the 3 and 4 positions of the phenyl group relative to theside chain.

In a second embodiment, the invention relates to a method of modifying asubstrate surface to provide a desired functionality, the methodcomprising contacting at least a portion of the substrate surface withan alkaline, aqueous solution under oxidative conditions, the solutioncomprising a SMA according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; wherein the substrate surface is modified; andcontacting the surface-modified substrate with a reactive moiety,wherein the reactive moiety reacts with and is bound to the modifiedsurface. The reactive moiety comprises nucleophiles or metals.

In a third embodiment, the invention provides a method of reducingamounts of metal in a fluid comprising the steps of contacting at leasta portion of a substrate with an alkaline, aqueous solution underoxidative conditions, the solution comprising a SMA according to FormulaI:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; contacting the surface-modified substrate with areactive moiety, wherein the reactive moiety reacts with and is bound tothe modified surface; and positioning the surface in a fluid with metal,whereby the modified surface binds at least a portion of the metal andwherein the reactive moiety is a metal.

In a fourth embodiment, the invention provides a method of modifying asubstrate surface to form a biofouling-resistant, modified substratesurface, the method comprising the steps of contacting at least aportion of the surface of the substrate surface with an alkalinesolution under oxidative conditions, the solution comprising a SMAaccording to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and contacting at least a portion of thesurface-modified substrate with a biofouling-resistant reactive moiety,wherein a biofouling-resistant, surface-modified substrate is formed. Inone embodiment, the surface-modified substrate is part of a medicaldevice, and the biofouling-resistant reactive moiety is selected fromthe group consisting of thiols, primary amines, secondary amines,nitriles, aldehydes, imidazoles, azides, halides, polyhexamethylenedithiocarbonate, hydrogen, hydroxyls, carboxylic acids, aldehydes,carboxylic esters or carboxamides.

In a fifth embodiment, the invention provides a kit for modifying asubstrate surface, the kit comprising a SMA according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and instructions for use. The surface-modifyingagent may be in solution or in powdered form.

The kit may further comprise a reactive moiety selected from the groupconsisting of thiols, primary amines, secondary amines, nitriles,aldehydes, imidazoles, azides, halides, polyhexamethylenedithiocarbonate, hydrogen, hydroxyls, carboxylic acids, aldehydes,carboxylic esters or carboxamides and a substrate surface to bemodified.

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . . ” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Mussel-inspired surface-independent adhesive. Photograph of amussel attached to commercial polytetrafluoroethylene (PTFE).

FIG. 1B. Schematic illustrations of the interfacial location of Mefp-5.

FIG. 1C. Schematic illustrations of the interfacial location of asimplified molecular representation of characteristic amine and catecholgroups.

FIG. 1D. The amino acid sequence of Mefp-5 (SEQ ID NO:1).

FIG. 1E. Amine and catechol functional groups of dopamine.

FIG. 1F. Schematic illustration of thin film deposition of polydopamineby dip-coating an object in an alkaline dopamine solution.

FIG. 1G. Thickness evolution of polydopamine coating on Si as measuredby AFM of patterned substrates.

FIG. 1H. XPS characterization of twenty-five differentpolydopamine-coated substrates. The bar graph represents the intensityof characteristic substrate signal before (hatched) and after (filled)coating by polydopamine. The intensity of the unmodified substratesignal is in each case normalized to 100%. Substrates withcharacteristic XPS signals indistinguishable from polydopamine aremarked by N.A. The circles represent the nitrogen-to-carbon ratio (N/C)after polydopamine coating.

FIG. 2A. Reaction Sscheme I of dopamine oxidation.

FIG. 2B. Reaction Scheme II of dopamine oxidation.

FIG. 3A. X-ray Photoelectron Spectroscopy (XPS) characterization ofpolydopamine-coated substrates. XPS spectral changes of gold (Au)palladium before (left column) and after (right column) polydopaminecoating.

FIG. 3B. XPS spectral changes of silver (Ag) palladium before (leftcolumn) and after (right column) polydopamine coating.

FIG. 3C. XPS spectral changes of palladium before (left column) andafter (right column) polydopamine coating.

FIG. 3D. XPS spectral changes of palladium before (left column) andafter (right column) polydopamine coating.

FIG. 3E. XPS spectral changes of copper before (left column) and after(right column) polydopamine coating.

FIG. 3F. XPS spectral changes of stainless steel before (left column)and after (right column) polydopamine coating.

FIG. 3G. XPS spectral changes of NiTi before (left column) and after(right column) polydopamine coating.

FIG. 3H. XPS spectral changes of titanium before (left column) and after(right column) polydopamine coating.

FIG. 3I. XPS spectral changes of silicon before (left column) and after(right column) polydopamine coating.

FIG. 3J. XPS spectral changes of quartz before (left column) and after(right column) polydopamine coating.

FIG. 3K. XPS spectral changes of, aluminum oxide before (left column)and after (right column) polydopamine coating.

FIG. 3L. XPS spectral changes of GaAs before (left column) and after(right column) polydopamine coating.

FIG. 3M. XPS spectral changes of Si₃N₄ before (left column) and after(right column) polydopamine coating.

FIG. 3N. XPS spectral changes of Nb₂O₅ before (left column) and after(right column) polydopamine coating.

FIG. 3O. XPS spectral changes of PS before (left column) and after(right column) polydopamine coating.

FIG. 3P. XPS spectral changes of PE before (left column) and after(right column) polydopamine coating.

FIG. 3Q. XPS spectral changes of PC before (left column) and after(right column) polydopamine coating.

FIG. 3R. XPS spectral changes of PET before (left column) and after(right column) polydopamine coating.

FIG. 3S. XPS spectral changes of PTFE before (left column) and after(right column) polydopamine coating.

FIG. 3T. XPS spectral changes of PDMS before (left column) and after(right column) polydopamine coating.

FIG. 3U. XPS spectral changes of PEEK before (left column) and after(right column) polydopamine coating.

FIG. 3V. XPS spectral changes of PU-1 before (left column) and after(right column) polydopamine coating.

FIG. 3W. XPS spectral changes of PU-2 before (left column) and after(right column) polydopamine coating.

FIG. 3X. XPS spectral changes of glass before (left column) and after(right column) polydopamine coating.

FIG. 3Y. XPS spectral changes of HAp) before (left column) and after(right column) polydopamine coating. The characteristic XPS substratesignals for unmodified substrates (left) were marked by filled circles,which were completely suppressed after polydopamine coating (right).Instead, carbon (−285 eV), nitrogen (399.5 eV), and oxygen (532.5 eV)photoelectron peaks (in order from low to high binding energy) wereobserved. The area ratio of nitrogen-to-carbon was determined fortwenty-five different substrates, and those values are shown in FIG. 1H(circles). Substrate XPS peaks used in the experiments are summarized inTable 2.

FIG. 4. Preliminary GPC analysis of dopamine solution after incubationfor 60 hours at room temperature. Mobile phase buffer: 50 mM sodiumphosphate, 100 mM NaCl, pH 6.5 with a flow rate of 0.3 mL/minutes (min).The sample was filtered before injection (pore size—0.8 microns (μm) andthe retention times of molecular weight standards are indicated by thearrows. The broad peak at a retention time (40 min) correlates topolydopamine at an approximate molecular weight of about several millionDalton based on molecular weight standards (PEG, 5 kDa, Bovine SerumAlbumin (BSA), 66 kDa, and polyacrylic acid (PAA), 1 MDa). A second peakat an elution time of 80 min indicates oligomer formation, and a thirdpeak found at the retention time of 95 min is due to a contaminant inthe GPC system.

FIG. 5A. Time of flight secondary ion mass spectrometry (ToF-SIMS)analysis of polydopamine coating, suggested reaction, and organicad-layer formation mechanisms. ToF-SIMS spectra of polydopamine-coatedglass. The mass spectrum showed a trimer of 5,6-dihydroxyindole,possibly fragmented from a long-chain polymer of similar composition.The characteristic pattern of fragmentation suggests liberation of twohydroxyl groups and a portion of the phenyl group, identifying eachsubunit as derived from dopamine polymerization.

FIG. 5B. Possible structural evolution and polymerization mechanisms ofdopamine, as well as suggested reaction mechanisms for organic ad-layerformation on polydopamine-coated substrates. Under an oxidativecondition (e.g. alkaline pH, oxidants, etc.) dihydroxyl group protons indopamine are deprotonated, becoming dopamine-quinone, which subsequentlyrearranges via intramolecular cyclization to leukodopaminechrome.Further oxidation and rearrangement leads to 5,6 dihydroxyindole, whosefurther oxidation causes inter-molecular cross-linking to yield apolymer that is structurally similar to the bio-pigment melanin. Thepolydopamine-coated substrate subsequently reacts with a variety ofmolecules via Shiff-base (top) and Michael addition chemistries.

FIG. 6A. Characterization of a surface-independent polydopamine-coatedsubstrate. The thickness evolution of a polydopamine-coated substrate asa function of dopamine coating time. Topological images of atomic forcemicroscopy (AFM) measured a height difference between substrate(silicon) and the polydopamine-coated layer generated byphotolithography. The bottom inset is 3D representation of actual AFMimaging of a 34 nanometers (nm) coating (6 hours (hr)). The top inset isthe cross-section of a 19 nm polydopamine-coating (3 hr).

FIG. 6B. ToF-SIMS on the polydopamine-coated substrate. The mass 445 m/zis the product of a fragmented polydopamine chain showing a trimer of5,6-dihydroxlindole and leukodopaminechrome resulting from dopamineoxidation (refer to FIG. 12). The dehydroxylation (α) followed by phenylring opening (β) indicates that a catechol moiety is the major componentin the SMA-treated substrate.

FIG. 7A. Polydopamine-assisted electroless metallization of substrates.Electroless copper deposition on polydopamine-coated nitrocellulosefilm.

FIG. 7B Electroless copper deposition on polydopamine-coated coin.

FIG. 7C. Electroless copper deposition on polydopamine-coated on athree-dimensional plastic object.

FIG. 7D. Schematic representation of electroless metallization ofphotoresist-patterned polydopamine-coated substrates. Photoresist wasremoved before silver metallization (left) or after copper metallization(right).

FIG. 7E. Scanning Electron Microscopy (SEM) images showing micropatternsof silver on Si.

FIG. 7F. Scanning Electron Microscopy (SEM) images showing micropatternsof silver on copper on a glass substrate.

FIG. 8A. XPS and ToF SIMS characterization of silver ad-layer depositedon polydopamine-coated substrates by electroless metallization. XPSspectra taken at each step of surface modification. (Top) Cleanunmodified silicon nitride exhibited Si (2p=101.5 eV), N (1s=397.5 eV),and 0 (1s=532.5 eV) peaks. (Middle) Polydopamine-coated silicon nitrideexhibited C, N, and 0 signals characteristic of polydopamine. (Bottom)The silver metal layer formed on polydopamine-coated silicon nitride,showing strong metallic Ag peaks (3d₅₁₂=368.6 eV; 3d₃₁₂=374.7 eV) andminor hydrocarbon contamination. Inset: Angle-dependent (60, 45, 30, and20 degrees from top to bottom). XPS showed no nitrogen is at take-offangles of 30 deg or less, confirming metallic silver ad-layer formationon top of polydopamine.

FIG. 8B. Electroless silver deposition on various substrates. Silver onglass (top left), gold (top right), Ti (bottom left), and PEEK (bottomright) showed nearly identical ToF-SIMS spectra in which two strongsilver isotope peaks at 106.8 (theoretical 106.9) and 108.8 (theoretical108.9) m/z were observed.

FIG. 9. ToF-SIMS characterization of copper ad-layer deposited byelectroless metallization onto diverse polydopamine-coated substrates.All ToF-SIMS mass spectra were similar regardless of underlyingsubstrates (62.9 and 64.9 m/z with an isotopic ratio of roughly 100%(62.9 m/z) to 40% (64.9 m/z)), indicating successful metallic copperdeposition in a substrate-independent manner. The peak at 23 m/z was Nacontamination.

FIG. 10. Roughness analysis of polydopamine- and metallizedpolydopamine-coated substrates. The root mean square (RMS) roughness ofNb₂O₅, polydopamine-coated Nb₂O₅, and Cu-polydopamine-coated Nb₂O₅ wasmeasured by AFM. The RMS roughness was 0.2 nm for bare Nb₂O₅ (left), 3.4nm for polydopamine-coated Nb₂O₅ (middle), and 31.7 nm forCu-polydopamine-coated Nb₂O₅ (right).

FIG. 11A. Copper metallization in the absence of polydopamine-coatedsubstrates. Copper metallization became inefficient without immobilizedcopper ions on polydopamine-coated silicon.

FIG. 11B. Copper metallization in the absence of polydopamine-coatedsubstrates. Copper metallization became inefficient without immobilizedcopper ions on polydopamine-coated niobium oxide.

FIG. 11C. Copper metallization in the absence of polydopamine-coatedsubstrates. Copper metallization became inefficient without immobilizedcopper ions on polydopamine-coated polytetrafluoroethylene (PTFE). Thesubstrates showed different preferences for copper metallization withoutthe polydopamine-coating. Metallic copper was barely detected on PTFEsimilar to unmodified substrates. Although the niobium oxide substrateexhibited good affinity to a copper layer, the niobium signalsindicating that the Cu layer was not perfect. Silicon wafer revealedpoor adhesion for newly formed metallic Cu layers showing a substrateSi2p signal.

FIG. 12A. Preparing surface-independent non-fouling (i.e., proteinresistant) substrates. Total internal reflection fluorescence (TIRF)microscope images of a protein adsorption at a single molecule level.Significant amount of the surface adsorption of fluorophore-labeledproteins onto the unmodified glass surface was shown after 30 min (top).Protein adsorption resistance by PEG (mPEG-NH₂, 5 kDa) conjugated onpolydopamine-coated glass substrates after continuous 30 min (middleleft) and 48 hr (middle right) exposure to proteins. A proposeddescription of the surface chemistry for the protein inert substratespreparation (top schematic). Positive control experiments (bottom).Glass substrates were PEGylated by the standard silane chemistry andsubsequent exposure to protein solutions for 30 min (bottom left) and 48hr (bottom right) showing a defective surface.

FIG. 12B. In vitro antifouling evaluation of various substrates (hatch).Short-term (4 hr) fibroblast adhesion test revealed significantlyimproved antifouling properties for all tested materials includingoxides, metals, semiconductors, and polymers (solid).

FIG. 12C. The XPS sulfur 2p (163 eV) signals on the polydopamine-coatedglass substrate indicates successful interfacial PEG immobilization.Inset shows the high-resolution spectrum of the sulfur 2p region markedby the arrow in a survey scan.

FIG. 13A. Polydopamine-assisted grafting of various organic moleculesonto polydopamine-coated substrates. Schematic illustration ofalkanethiol monolayer (top right) and PEG polymer (bottom right) graftedon polydopamine-coated substrates.

FIG. 13B. Pictures of water droplets on several unmodified (left),polydopamine-coated (middle), and alkanethiol-grafted substrates(right). Substrates investigated include organic polymers (PTFE, PC, andnitrocellulose (NC)), metal oxides (SiO₂ and TiO₂), and noble metals (Cuand Au). Contact angle values are shown in Table 3.

FIG. 13C. NIH 3T3 fibroblast cell adhesion to unmodified glass andOEG6-terminated alkanethiol monolayer formed on polydopamine-coatedglass.

FIG. 13D. TIRF microscopy of Cy3 conjugated Enigma homolog proteinadsorption to mPEG-NH₂-grafted polydopamine-coated glass (48 hr exposureto protein solution).

FIG. 13E. TIRF microscopy of Cy3 conjugated Enigma homolog proteinadsorption to bare glass (30 min exposure).

FIG. 13F. TIRF microscopy of Cy3 conjugated Enigma homolog proteinadsorption to PEG-silane immobilized on bare glass (48 hr exposure).

FIG. 13G. NIH 3T3 fibroblast cell adhesion to polydopamine-coatedsubstrates after grafting with mPEG-SH (Pre-normalized data areavailable in Table 4).

FIG. 14. XPS analysis of self-assembled monolayer formed onpolydopamine-coated polycarbonate. XPS survey spectrum after reactionbetween dodecanethiol and polydopamine-coated polycarbonate. Arrowrepresents the sulfur 2p (163 eV) signal derived from the surfaceimmobilized dodecanethiol molecules. Inset shows the high-resolutionspectrum of the sulfur 2p region marked by the arrow.

FIG. 15. XPS analysis of PEG grafted onto polydopamine-coated glass. XPSsurvey spectrum after reaction between mPEG-SH and polydopamine-coatedglass. Arrow represents the sulfur 2p (163 eV) signal derived from thesurface-immobilized mPEG-SH molecules. Inset shows the high-resolutionspectrum of the sulfur 2p region marked by the arrow.

FIG. 16A. Chemically ‘active’ coating targeting cellular proteins forthe study of bacterial chemotaxis. Experimental scheme. Thepolydopamine-coated latex micro-bead was chemically conjugated to an E.coli flagella protein which enabled real-time monitoring of rotation offlagellum.

FIG. 16B. Real-time images of counterclockwise rotation of singleflagellum with a time resolution of 50 msec (starting from upper left).Spatially confined moving traces of the attached bead showedcounterclockwise rotation of the flagellum. Notation of flagellarotational direction is opposite to the normal usage: flagellum rotates‘clockwise’ from experimenter's point of view in this figure, whichshould be expressed as ‘counterclockwise’ rotation in bacterialchemotaxis. The rotational direction is determined from the bacterialpoint of view.

FIG. 17A. Polydopamine-assisted grafting of a biomacromolecule forbiospecific cell interaction. Representative scheme for hyaluronic acid(HA) conjugation to polydopamine-coated substrates.

FIG. 17B. Adhesion of M07e cells on polydopamine-coated polystyrene (PS)increases with the HA solution concentration used during grafting.

FIG. 17C. Bioactive HA ad-layers were formed on polydopamine-coatedglass, tissue-culture PS, and indium tin oxide (ITO), as demonstrated byattachment of M07e cells. Competition with soluble HA (bar at the rightend, PS+sol HA) confirmed that cell adhesion was due to grafted HA.

FIG. 17D. Polydopamine-modified PS grafted with HA (0.5 mg/mL) retainsbioactivity during long-term culture with M07e cells. Images taken afternormal-force centrifugation show almost 100% attachment of expandingM07e cells at day 2 (FIG. 17D; 2760±390 cells/cm²).

FIG. 17E. Polydopamine-modified PS grafted with HA (0.5 mg/mL) retainsbioactivity during long-term culture with M07e cells. Images taken afternormal-force centrifugation show almost 100% attachment of expandingM07e cells at days 4 (FIG. 17E; 5940±660 cells/cm²).

FIG. 17F. In the absence of HA, the polydopamine-coated substratessupported similar levels of M07e cell expansion at day 4, but did notsupport cell adhesion (610±630 cells/cm²).

FIG. 18A. Flow cytometry analysis of CD44 levels on M07e cells. M07ecells were stained with isotype control-APC.

FIG. 18B. Flow cytometry analysis of CD44 levels on M07e cells. M07ecells were stained with r anti-CD44-APC antibodies to determine thesurface expression of CD44 receptors. The fraction of cells expressingCD44 was determined by quantifying the number of cells within the samplehaving fluorescence intensity greater than isotype-control-stained cells(P2=99.4% for CD44-APC stained cells). Data are representative of twoindependent experiments.

FIG. 19. M07e cell expansion on TCPS, polydopamine, and HA-polydopaminesubstrates. Similar cell expansion was observed on all three substrates.Curves are best-fit exponential and error bars show standard deviation.Represents average of thirteen experiments/timepoints.

FIG. 20A. High resolution x-ray photoelectron spectroscopy (XPS)analysis of C1s region of polydopamine-coated SiOx substrate aftersecondary reaction of pHis at pH 4.0 (1) and 6.8 (2).

FIG. 20B. High resolution x-ray photoelectron spectroscopy (XPS)analysis of O1s of polydopamine-coated SiOx substrate after secondaryreaction of pHis at pH 4.0 (1) and 6.8 (2).

FIG. 21A. ToF-SIMS analysis of pHis-polydopamine-coated siliconsubstrate.

FIG. 21B. P ToF-SIMS analysis of olydopamine-coated substrate.

FIG. 22A. Hetero-bifunctional Ac—N-His-OEG₃-Lys.

FIG. 22B. Matrix Assisted Laser Desorption/Ionization Time of Flightmass spectrometry (MALDI-ToF MS) spectra after synthesis andpurification of Ac—N-His-OEG₃-Lys.

FIG. 23A. Peroxidase activities monitored at 420 nm as a function of pH.

FIG. 23B. Polydopamine-His configuration allowed biotinylation whichserves as a platform for streptavidin-peroxidase immobilization at mildacidic and neutral pHs.

FIG. 23C. Polydopamine-Lys orientation preventedbiotinylation/streptavidin-peroxidase immobilization, resulting in lowperoxidase activities at alkaline pHs

FIG. 24. Different conditions (pH and concentration of dopamine) forpolydopamine coating on polystyrene substrates.

FIG. 25. Contact angle of each substrate was measured before (solid) andafter (hatch) norepinephrine coating. The contact angle after coatingwas relatively consistent, indicating successful norepinephrine coating,whereas the contact angles of bare materials varied from hydrophilic(approximately 10°) to hydrophobic (approximately 130°).

FIG. 26A. XPS data to determine SMA-treatment on untreated substratesPC.

FIG. 26B. XPS data to determine SMA-treatment on PEEK.

DETAILED DESCRIPTION OF THE INVENTION I. In General

The present invention provides a novel, surface-independent,surface-modification method whereby substrates of all kinds are modifiedto support at least one functional ad-layer on the substrate's surface.In general, the method comprises contacting at least a portion of thesubstrate with a surface-modifying agent (SMA) to provide a surfacemodified to support at least one reactive moiety. The presentinvention's interfacial chemistry will be useful in important fieldsincluding biocompatible coatings of medical devices, surfacemodifications of drug delivery carriers and tissue engineeringscaffolds, biosensors, biofouling-resistant, industrial and consumercoatings, semiconductors, metal removal, surface catalysts and nextgeneration electronic displays.

The method comprises contacting at least a portion of a substrate withan alkaline solution under oxidizing conditions, the solution comprisinga SMA according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and wherein the substrate surface is modified.

After contact with the Formula I solution, the substrate surface ismodified. In a preferred embodiment the substrate surface is modified tocomprise a polymeric coating. The SMA-treated surface may then becontacted with a reactive moiety to provide a SMA-treated surface havinga functional ad-layer. The ad-layer can be tailored for specificapplications and may include one or more ad-layers. For instance, in oneembodiment, the SMA-treated surface may be modified to provide anad-layer comprising at least one reactive moiety such as metals,nucleophiles and polymers.

The following paragraphs enumerated consecutively from 1 through 31provide for various aspects of the present invention. In one embodiment,in a first paragraph (1), the present invention pertains to a method ofmodifying a substrate surface, the method comprising contacting at leasta portion of the substrate with an alkaline solution under oxidativeconditions, the solution comprising a SMA according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10; wherein y ranges from 0 to 10, provided that x or yis at least 1; and wherein the substrate surface is modified.

In a second paragraph (2), the invention relates to the method ofparagraph 1, wherein x and y are both 1, and where R₁ and R₄ form adouble bond when eliminated.

In a third paragraph (3), the invention relates to the method of eitherof paragraphs 1 or 2, wherein R₂ is NH₂ or NHR, wherein R is an alkyl oraromatic group.

In a fourth paragraph (4), the invention relates to the method of any ofparagraphs 1 through 3, wherein one of R₁ or R₄ is a halide, a hydroxylor a thiol and one of R₃ or R₅ is a hydrogen atom.

In a fifth paragraph (5), the invention relates to the method ofparagraph 1, wherein x is 1, y is 1, R₁ is a hydroxyl and R₃, R₄ and R₅are a hydrogen.

In a sixth paragraph (6), the invention relates to the method ofparagraphs 4 and 5, wherein R₂ is a NH₂.

In a seventh paragraph (7), the invention relates to the method ofparagraph 1, wherein x and y are each 1 and each of R₁, R₃, R₄ and R₅are hydrogen atoms.

In an eighth paragraph (8), the invention relates to the method ofparagraph 7, wherein R₂ is NH₂.

In a ninth paragraph (9), the invention relates to the method ofparagraph 1, wherein R₂ is NH₂ or NHR, wherein R is an alkyl or aromaticgroup

In a tenth paragraph (10), the invention relates to the method of any ofparagraphs 1 through 9 wherein one of R₁ or R₄ is a halide, a hydroxylor a thiol and one of R₃ or R₅ is a hydrogen atom.

In an eleventh paragraph (11), the invention relates to the method ofeither of paragraphs 9 or 10, wherein R₂ is an amine.

In a twelfth paragraph (12), the invention relates to the method of anyof paragraphs 1 through 11, wherein x+y is at least 2.

In a thirteenth paragraph (13), the invention relates to the method ofany of paragraphs 1 through 12, wherein x+y is at least 3.

In a fourteenth paragraph (14), the invention relates to the method ofany of paragraphs 1 through 13 wherein the hydroxyls of the phenylmoiety are positioned at the 3 and 4 positions of the phenyl grouprelative to the side chain.

In a fifteenth paragraph (15), the invention relates to the method ofany of paragraphs 1 through 14, wherein Formula I forms a polymeric coaton the substrate surface.

In a sixteenth paragraph (16), the invention relates to the method ofparagraph 1 wherein the surface-modifying agent is selected from thegroup consisting of 3,4-dihydroxy-L-phenylalanine (DOPA),3,4-dihydroxyphenylalanine methyl ester, dopamine, norepinephrine,epinephrine and salts thereof.

In a seventeenth paragraph (17), the invention relates to the method ofany of paragraphs 1 through 16 wherein the solution is aqueous.

In an eighteenth paragraph (18), the invention relates to the method ofany of paragraphs 1 through 17 wherein x+y ranges from 1 to 6.

In a nineteenth paragraph (19), the invention relates to a method ofmodifying a substrate surface to provide a desired functionality, themethod comprising contacting at least a portion of the substrate surfacewith an alkaline, aqueous solution under oxidative conditions, thesolution comprising a surface-modifying agent according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and wherein the substrate surface is modified; andcontacting the surface-modified substrate with a reactive moiety,wherein the reactive moiety reacts with and is bound to the modifiedsurface.

In a twentieth paragraph (20), the invention relates to the method ofparagraph 19, wherein the reactive moiety comprises a nucleophile.

In a twenty-first paragraph (21), the invention relates to the method ofparagraph 19 wherein the reactive moiety comprises a metal.

In a twenty-second paragraph (22), the invention relates to a method ofreducing amounts of metal in a fluid comprising the steps of contactingat least a portion of a substrate with an alkaline, aqueous solutionunder oxidative conditions, the solution comprising a SMA according toFormula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; contacting the surface-modified substrate with areactive moiety, wherein the reactive moiety reacts with and is bound tothe modified surface; and positioning the surface in a fluid with metal,whereby the modified surface binds at least a portion of the metal.

In a twenty-third paragraph (23), the invention relates to the method ofparagraph 22 wherein the reactive moiety is a metal.

In a twenty-fourth paragraph (24), the invention relates to a method ofmodifying a substrate surface to form a biofouling-resistant, modifiedsubstrate, the method comprising the steps of contacting at least aportion of the substrate surface with an alkaline solution underoxidative conditions, the solution comprising a SMA according to FormulaI:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and contacting at least a portion of thesurface-modified substrate with a biofouling-resistant reactive moiety,wherein a biofouling-resistant, modified substrate surface is formed.

In a twenty-fifth paragraph (25), the invention relates to the method ofparagraph 24 wherein the biofouling-resistant reactive moiety isselected from the group consisting of thiols, primary amines, secondaryamines, nitriles, aldehydes, imidazoles, azides, halides,polyhexamethylene dithiocarbonate, hydrogen, hydroxyls, carboxylicacids, aldehydes, carboxylic esters or carboxamides.

In a twenty-sixth paragraph (26), the invention relates to the method ofparagraphs 24 and 25 wherein the modified substrate surface is part of amedical device.

In a twenty-seventh paragraph (27), the invention relates to a kit formodifying a substrate surface, the kit comprising a SMA according toFormula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and instructions for use.

In a twenty-eighth paragraph (28), the invention relates to the kit ofparagraph 27 further comprising a reactive moiety selected from thegroup consisting of thiols, primary amines, secondary amines, nitriles,aldehydes, imidazoles, azides, halides, polyhexamethylenedithiocarbonate, hydrogen, hydroxyls, carboxylic acids, aldehydes,carboxylic esters or carboxamides.

In a twenty-ninth paragraph (29), the invention relates to the kit ofparagraphs 27 and 28 further comprising a substrate surface to bemodified.

In a thirtieth paragraph (30), the invention relates to the kit ofparagraphs 27 through 29, wherein the surface-modifying agent is insolution.

In a thirty-first paragraph (31), the invention relates to the kit ofparagraphs 27 through 29, wherein the surface-modifying agent is inpowdered form.

These embodiments are described in more detail below.

II. Surface-Modifying Agents (SMAs) Formula I.

In a preferred embodiment, the substrate surface is modified bycontacting at least a portion of the substrate with a dilute, alkalinesolution under oxidizing conditions, the solution comprising a SMAaccording to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and wherein the substrate surface is modified.

Dopamine.

In another preferred embodiment, the substrate surface is modified bycontacting at least a portion of the substrate with a SMA wherein theSMA is dopamine or dopamine salts:

Substrates treated with dopamine are reactive with organic heteroatomssuch as amine and thiol groups by Schiff base or Michael additionreactions (pathway “b,” “c” reaction sequence II-FIG. 2B) and alsostrongly binds to various metals such as Fe, Cu, Hg, and Zn (pathway “a”of reaction sequence II-FIG. 2B). Thus our new concept ofsurface-independent, surface-modifying chemistry emerges: theself-polymerized multilayer nanofilm of dopamine providesmulti-functionality due to chemical reactions or metal bindings at a toplayer of a solid-liquid or solid-vapor interface whereas the bottomlayer is attached to versatile organic and inorganic substrates. Thesevery unique; current interface modifiers require chemical synthesisincorporating chemical orthogonality at each end.

In one preferred embodiment dopamine is used. When a substrate iscontacted with dopamine, an adherent polydopamine polymeric film iscoated on the substrate. Dopamine oxidation chemistry may be summarizedby reaction sequence (I) in FIG. 2A. There, dopamine's dihydroxyl groupsare deprotonated under oxidative conditions (neutral or alkaline),becoming dopamine-quinone. Rearrangement results in intra-molecularcyclization which reproduces dihydroxyl groups from quinone.

The second oxidation generates dopamine-chrome which quickly rearrangesto form a stable phenyl ring structure creating an additional doublebond in the 5-membered ring (5,6-dihydroxyindole). The third oxidationstarts inter-molecular cross-linking due to the full unsaturated natureof indole forming polymer both in solution and on the substrate.

Norepinephrine.

In another preferred embodiment, the substrate surface is modified bycontacting at least a portion of the substrate with a SMA wherein theSMA is norepinephrine or norepinephrine salts:

Norepinephrine is a neurotransmitter found in the brain which has anadditional hydroxyl group in the carbon spacer of dopamine.

Other preferred SMAs include 3,4-dihydroxy-L-phenylalanine (DOPA),3,4-dihydroxy-L-phenylalanine methyl ester, epinephrine and saltsthereof.

The alkaline solution of SMA of the present invention can also includeadditives such as fillers, pigments, wetting agents, viscositymodifiers, stabilizers, anti-oxidants or cross-linking agents. The SMAcan be cross-linked if desired. If desired, the SMA solution can includevarious adjuvants such as small particle fillers, surface active agents,UV absorbers, photo-initiators, colorants and indicators.

The surface-independent, surface-modification method of the presentinvention comprises contacting at least a portion of the substratesurface with a SMA under oxidative conditions to form a surface-modifiedsubstrate surface having at least one reactive moiety on the substrate'ssurface. The method comprises contacting at least a portion of thesubstrate with an alkaline solution under oxidizing conditions, thesolution comprising a SMA according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and wherein the substrate surface is modified.

By “dilute,” we mean that the concentration of SMA is 0.01 mg/ml-100mg/ml, preferably ranging from about 0.05 mg/ml or higher.

By “alkaline,” we mean that the pH value of the solution ranges from 7.1to 12, with a preferred pH ranging from 7.5 to 10, with a furtherpreferred pH ranging from 7.5 to 8.5. An alkaline solution triggerspolymerization of SMAs onto the substrate surface.

By “solution,” we mean both aqueous and non-aqueous solvents, includingmiscible solutions of water and organic solvents such as acetone,chloroform, dichloromethane, methanol, ethanol, isopropanol,dimethylformamide, dimethylsulfoxide and hexane. Preferably, thesolution is made just prior to contacting the substrate, although thesolution may be stored for at least brief periods of time before use.

By “under oxidative conditions,” we mean alkaline pH of aqueoussolutions and non-aqueous solvents with dissolved oxygen or organicbases such as triethylamine. In alternative embodiments, solutionscomprising oxidants such as hydrogen peroxide, sodium periodate,tertiary butylhydroperoxide, organic peroxides, quinones includingbenzoquinones, napthoquinones, anthraquinones, nitroaryl compounds,metal oxidants including Cu²⁺, Fe³⁺, Co³⁺ and Mn³⁺, phenols, indoles,aminobenzenes and more can be used to initiate polymerization viaoxidization of the SMA.

By “contacting,” we mean exposing at least a portion of the substrate tothe SMA for a period of time ranging from 1 minute to 24 hours and arange of temperatures from 0° C. to 100° C. In a preferred embodiment,the substrate is exposed to the SMA for a period of time ranging from 2hrs to 18 hrs, preferably for 5 hrs to 15 hrs, even more preferably for8 hrs to 12 hrs.

In a preferred embodiment the entire substrate is immersed or dipped inthe SMA solution. The examples below illustrate preferred contactingmethods. However, a variety of techniques can be employed to contact thesubstrate surface with the SMA solution including, without limitation,swabbing, dip coating, spin coating, die coating, ink jet coating,spraying, screen printing (e.g., rotary screen printing), gravureprinting, photolithographic printing and flexographic printing,microcontact printing, nanolithography.

On contact, the substrate surface is preferably modified so as toprovide a substrate surface having at least one reactive moiety. In apreferred embodiment, the reactive moiety comprises a smooth, continuouspolymeric coating on the substrate surface, the polymeric coating havinga substantially constant thickness. As a general guide, the polymericcoating exists on the substrate surface in a thickness ranging fromabout 1 to 1000 nnm, preferably ranging from about 1 to 100 nm, morepreferably ranging from about 5 to 50 nm, and even more preferablyranging from about 10 to 50 nm.

IV. Substrates

The method comprises contacting at least a portion of the substrate withthe SMA described above.

By “substrate,” we mean any inorganic or organic substrate. Forinstance, the substrate can be an organic solid, an inorganic solid, ora combination of organic and inorganic solids that provides a surfacefor receiving the adherent polymer. Suitable organic or inorganicsubstrates may also be fibrous, filamentous, meshes, porous orsolvent-swollen (e.g. hydrogel or organogel) objects. Preferably, careis taken when selecting the substrate so that there will be an adequatedegree of adhesion between the substrate and the SMA.

Suitable inorganic substrates include but are not limited to inorganicsubstrates such as quartz, glass, silica and other oxides or ceramicssuch as alumina, indium tin oxide, lithium tantalate (LiTaO3), lithiumniobate (LiNbO3), gallium arsenide (GaAs), silicon carbide (SiC),langasite (LGS), zinc oxide (ZnO), aluminum nitride (AlN), aluminumoxide (Al₂O₃), silicon (Si), silicon nitride (Si3N4), and lead zirconiumtitanate (“PZT”), titanium oxide (TiO₂), niobium oxide (Nb₂O₅); andmetals or alloys such as aluminum, copper, gold, silver and steel. Othersuitable inorganic substrates include, without limitation, mica, diamondand nickel titanium (NiTi).

Suitable organic substrates include but are not limited to organicsubstrates such as thermoplastics including polyesters (e.g.,polyethylene terephthalate or polyethylene naphthalates), polyacrylates(e.g., polymethyl methacrylate or “PMMA”), poly(vinyl acetate) (“PVAC”),poly(vinylbutyral) (“PVB”), poly(ethyl acrylate) (“PEA”),poly(diphenoxyphosphazene) (“PDPP”), polycarbonate (“PC”), polypropylene(“PP”), high density polyethylene (“HDPE”), low density polyethylene(“LDPE”), polysulfone (“PS”), polyether sulfone (“PES”), polyurethane(“PUR”), polyamide (“PA”), poly(dimethylsiloxane) (“PDMS”), polyvinylchloride (“PVC”), polyvinylidene fluoride (“PVdF”), polystyrene (“PSy”)and polyethylene sulfide; and thermoset plastics such as cellulosederivatives, polyimide, polyimide benzoxazole and polybenzoxazole. Othersuitable organic substrates include, without limitation, graphite,carbon nanotubes, fullerenes, graphene, poly(glycolic acid), poly(lacticacid), and poly(lactic-co-glycolic acid) and Teflon®.

Untreated Substrates.

The method of the present invention can be used on substrates in anycondition (see Examples 2 and 3). For instance, substrates havingexisting coatings such as paint, oil, grease, protectants and the likecan be used without any additional pre-treatments or cleaning.

Pre-Treated Substrates.

In another embodiment, the substrate can instead or in addition to bepretreated to enhance surface-modification. Preferred pretreatmentsinclude but are not limited to electron and ion beam irradiation,electrical discharge in the presence of a suitable reactive ornon-reactive atmosphere (e.g., plasma, glow discharge, corona discharge,dielectric barrier discharge or atmospheric pressure discharge);chemical pretreatment (e.g., with a low solids solution ofpolyvinylidene dichloride or with a solvent-borne mixture of a polyesterresin and an aziridine cross-linker); flame pretreatment; ultravioletlight pretreatment with or without ozone pretreatment; and incorporatingfunctional polymers into the substrate when a polymeric substrate isemployed. In an alternative embodiment, the present invention provides amethod of enhancing coatings on artificially or naturallydamaged/altered substrates.

V. Reactive Moiety

The surface-independent, surface-modifying biocoating of the presentinvention provides an amazingly versatile platform for secondaryreactions, allowing one to tailor specific reactive moieties tosubstrates for diverse functional uses. For instance, the SMA-treatedsubstrates of the present invention are conformal and chemicallyreactive with a wide variety of organic and inorganic species such asmetal ions, thiols, primary amines, secondary amines, nitriles,aldehydes, imidazoles, azides, halides, polyhexamethylenedithiocarbonate, hydrogen, hydroxyls, carboxylic acids, aldehydes,carboxylic esters or carboxamides. Thus, secondary reactions between theSMA-treated substrates and such reactive moieties can be exploited toimpart specific functionalities to the surface-modified substrate.

The oxidative pathways for adding the secondary reactions are set forthin reaction sequence II in FIG. 2B. There, pathway ‘a’ representsvarious metal bindings of dopamine. The metal ‘M’ can be titanium (Ti),iron (Fe), copper (Cu), zinc (Zn), silver (Ag), or many others. Pathway‘b’ is Schiff base and ‘c’ is Michael addition reactions which were usedfor the interfacial reactions with PEG-amine, PEG-thiol, and proteins(flagella).

Current applications for SMA-treated substrates having a reactive moietyare many and include, without limitation, applications foranti-biofouling surfaces; medical devices for catheters, stents,artificial bones, teeth, and dialysis tubes; semiconductors forbio-MEMS, and sensors; and metal nanoparticles and quantum dots forsensors, diagnostics, and cellular imaging.

Thus, in an alternate embodiment of the invention, a method of applyinga reactive moiety to a SMA-treated substrate is provided. The methodcomprises contacting at least a portion of a substrate with an alkalinesolution under oxidizing conditions, the solution comprising a SMA toform a substrate having a modified surface and then contacting theSMA-treated substrate with a reactive moiety to form a functionalad-layer on the SMA-treated substrate.

By “reactive moiety” we mean to include any reactive moiety includingmetals, nucleophiles and polymers. Specifically, we include thiols,primary amines, secondary amines, nitriles, aldehydes, imidazoles,azides, halides, polyhexamethylene dithiocarbonate, hydrogen, hydroxyls,carboxylic acids, aldehydes, carboxylic esters or carboxamides.

By “ad-layer” we mean an additional layer of reactive compounds whichbinds to the modified surface of the SMA-treated substrate and altersthe functionality of the substrate.

Electroless Metallization.

In this embodiment, one would preferably treat a surface with an SMA asdescribed above and then expose the treated surface to metal solutionsto form an adherent metal film. Example 5, below, describes thedip-coating of an SMA-treated substrate in a silver nitrate and copper(II) chloride solution. In general, one would wish to expose theSMA-treated substrate to a solution of 10-500 mM metal, pH 3-8, and20-70° C.

Nucleophile Addition.

In this embodiment, one would preferably contact a substrate with an SMAas described above and then expose the SMA-treated substrate tonucleophile. By “nucleophile” we mean an electron-rich species with atendency to be attracted to the nuclear charge of an electron-poorspecies, the electrophile. Important nucleophiles include primary andsecondary amines, thiols, azides, nitriles, aldehydes, imidazoles,azides, polyhexamethylene dithiocarbonate, hydrogen, hydroxyls,carboxylic acids, aldehydes, carboxylic esters or carboxamides, etc.

A partial list of important nucleophiles can be seen in Table 1:

TABLE 1 C1⁻ Br⁻ I⁻ HO⁻ *R—OH *RO⁻ H₂S HS⁻ *R—SH *R—S⁻ —NH₂ N₃ ⁻ ⁻C≡N*R—C≡C⁻ *R can be anything.

Suitable nucleophiles may comprise parts of more complex molecules, suchas proteins or nucleic acids. For instance, Example 9 describes labelingsurfaces with flagella. Example 10 describes fibroblast adhesion tosurfaces, Example 11 describes adding hyaluronic acid to surfaces andExample 13 describes addition of histidine to surfaces. In general,macromolecules containing the nucleophiles described above react toSMA-treated substrates.

Polymer Grafting.

In this embodiment, one would preferably contact a substrate with an SMAas described above and then expose the SMA-treated substrate to polymersincluding any synthetic polymers that contain nucleophiles as describedabove. For example, in the case of poly(ethylene glycol) (PEG),NH₂-PEG-NH₂, methoxy-PEG-NH₂, methoxy-PEG-SH, SH-PEG-SH,branched-PEG-NH2, and branched-PEG-SH are the polymer structuresreacting to SMA-treated surfaces. For instance, Example 8 describesgrafting PEG to SMA-treated surfaces. However, alternative forms ofpolymeric grafting are also envisioned, including free radical graftpolymerization, atom-transfer radical polymerization, plasmapolymerization/deposition, plasma treatment and surface irradiation, andcationic and anionic monomer or oligomer additions.

Metal Scavenging.

In this embodiment, the amount of metal ions in a fluid can be reducedby binding to SMA-treated substrates. By “reducing” we mean anyreduction in the amount of metal ions in solution, preferably to belowmaximum contaminant levels (MCL) or other established benchmarks for allmetals. The method comprises contacting at least a portion of asubstrate with an alkaline, aqueous solution comprising Formula I. Onethen positions the surface-modified substrate in a solution with metal,whereby the surface-modified substrate reduces the amount of metal inthe solution. The method can be performed in either flow-through orbatch mode. See Example 12 below for a preferred example.

VI. Kits

In an alternate embodiment of the invention, a kit for modifying asubstrate's surface is provided. In one embodiment, the kit comprises adilute, alkaline solution comprising a SMA according to Formula I, and,optionally, a substrate to be modified, and instructions for use. In apreferred embodiment, the kit comprises a powdered form of at least oneSMA, wherein the powdered SMA is hydrated by the user and for immediatecontacting with the substrate. For example, dopamine powder can beprovided for dissolving in a provided alkaline solution.

In an alternate embodiment, the kit comprises an SMA formulated,delivered and stored as a liquid in a nonoxidizing condition, forexample at a low pH. In this case the user would neutralize the liquidSMA to pH>7 for subsequent contacting with the substrate. For example,dopamine dissolved in acidic water can be provided for users to add base(NaOH) and substrates for coating.

In another alternative embodiment, a reactive moiety is also included,wherein a user can modify the SMA-treated substrate to include areactive moiety.

By “substrate” we mean any substrate described above, including anysubstrate wherein having at least one reactive moiety on the surfacewould be useful.

By “instructions for use” we mean a publication, a recording, a diagram,or any other medium of expression which is used to communicate theusefulness of the invention for one of the purposes set forth herein.The instructional material of the kit can, for example, be affixed to acontainer which contains the present invention or be shipped togetherwith a container which contains the invention. Alternatively, theinstructional material can be shipped separately from the container orprovided on an electronically accessible form on a internet website withthe intention that the instructional material and the SMA solution andsubstrate be used cooperatively by the recipient.

VII. Applications for SMA-Treated Substrates

Photolithography.

SMA-treated substrates can be used for subsequent photolithographymicropatterning and photoresist etching. Photolithography is a processused to selectively apply very precise geometric patterns ontosubstrates. Typically only very clean, flat substrates can be used forphotolithography. However, SMA-treated substrates allow virtually anysubstrate to be modified for use with photolithography, greatlyexpanding the types of materials which can be used in applicationsrequiring very small, very precise patterns including drug deliverycarriers, micro- and nano-wires for photonics, peptide arrays, proteinarrays, oligonucleotide arrays, electronic circuitry and integratedelectronic chips, electronic displays and the like.

SMA-Assisted Electroless Metallization.

SMA-treated substrates can be used to accept adherent and uniform metalcoatings by electroless metallization. Metals are often used assynthetic catalysts for chemical reactions with accelerated turn-overrates, such as platinum catalysts used to facilitate reactions ofaromatic conversion or branched isomers from straight alkane chains.Copper layers on substrates such as metals, semiconductors, and polymersare important for various electronic and packaging technologies,particularly in copper deposition on synthetic organic substrates forflexible printed circuit, electromagnetic interference shielding ofdisplay panels, and multichip module packing. However, currentapproaches can be applied for only one or a few types of substrates andoften involve complicated multi-step procedures. The present inventiontherefore describes a method of modifying the surface of any substrateto include a metal coating for use as, among other things, syntheticcatalysts, semiconductors, display panels, surface-metallization ofcantilever- or beam-based sensor devices, and carbon nanotubes.

Further, SMA-treated substrates can be used to accept electroless metaldeposition combined with conventional lithography processes to yieldmicropatterned metal-deposition on SMA-treated substrates. This providesan aqueous, cost-effective and surface-independent preparation processthat does not require toxic Pd/Sn colloids for catalysis, yieldingsubstrates useful in, for example, electronic circuit fabrication.

Biofouling.

SMA-treated substrates can be used to provide biofouling-resistantsubstrate for use in medical and dental devices and implants, watercrafthulls, off-shore and on-shore structures of manmade or naturalcomposition, water treatment facilities, liquid handling or movementstructures such as pipelines and chemical treatment facilities, foodprocessing surfaces, and construction and housing materials. By“biofouling” we mean the nonspecific adsorptions of macromolecules,cells, proteins, bacteria, algae and other organisms and theirbyproducts at solid-liquid or solid-air interfaces, often resulting inadverse effects on performance, safety, and longevity of, for instance,medical devices and sensors. By “resistant” we mean substrates modifiedso as to prevent the nonspecific adsorptions of macromolecules, cells,proteins, bacteria, algae and other organisms and their byproducts atsolid-liquid or solid-air interfaces associated with biofouling.Currently, surface immobilization of polyethylene glycol (PEG orPEGylation) has been the most popular approach for non-fouling surfacepreparation, but anchoring PEG molecules in a surface independent mannerremains a major challenge.

Biosensors.

SMA-treated substrates can be used to immobilize proteins and DNA onsubstrates for use in diagnosis, therapy of disease and experimentaltools for research in tissue and cellular proteomics and genomics.Immobilizing proteins and DNAs on substrates has revolutionizedthroughput of medical diagnostics and biological research for libraryscreening and gene expressions. So called protein and DNA chips requirechemical conjugations of biomacromolecules (DNA and proteins) ontosubstrates. Glass has dominated in this area due to its opticaltransparency and low cost. However, efforts have been made to developbioconjugate chemistry onto portable substrates such as paper forconvenient diagnostic purposes. Thus the versatile SMA-treatedsubstrates and method thereof presented herein can be applied to developportable diagnostic kits including biosensors, functional genomics,proteomics, and metabolomics, or hospital/clinic-base diagnostic devicesor their components.

Self-Assembled Monolayers.

SMA-treated substrates can be used to support a variety of reactionswith organic species for creating functional organic ad-layers. Forexample, under oxidizing conditions, catechols react with thiols andamines via Michael addition or Schiff base reactions (FIG. 5B). Thus,immersing SMA-treated substrates into a thiol- or amine-containingsolution provides a convenient route to organic ad-layer depositionthrough thiol- and amine-catechol adduct formation (FIG. 13A). Thefollowing examples demonstrate methods for depositing organic ad-layersin the form of alkanethiol monolayers, synthetic polymers, andbiopolymer coatings.

Polymeric Grafting.

SMA-treated substrates can be used to support polymeric ad-layers,including PEG, hyalurinic acid (HA), polyethylenimine, heparine,chitosan, or any other moiety described above. For example, PEG-grafted,SMA-treated substrates can be used for fouling-resistant substrates, andHA-immobilized surface is useful in hematopoietic cell cultures. Polymerad-layers were grafted onto SMA-treated substrates in a method accordingto the present invention, wherein the secondary reactive moietycomprises thiol- or amine-functionalized polymers, thus yieldingbioresistant and/or biointeractive substrates. Alternative forms ofpolymeric grafting are also envisioned, including free radical graftpolymerization, atom-transfer radical polymerization, plasmapolymerization/deposition, plasma treatment and surface irradiation, andcationic and anionic monomer or oligomer additions.

Protein Labeling.

SMA-treated substrates can be used to support protein ad-layers such asflagella, antibodies for diagnostic devices as well as therapeuticproteins and peptides for therapeutic purposes. For instance,flagella-labeled substrates are useful in chemotaxis and cellularnetwork studies. Currently, the only approach for singleflagella-labeling has been the physical adsorption of flagella antibodyon micro-bead substrates and subsequent incubation in the presence ofbacteria. By taking advantage of the chemical reactivity of SMA-treatedsubstrates to flagella proteins, a general route forbacteria-independent flagella labeling is proposed, thereby providing auseful labeling technique for research in areas such as food science,bacterial chemotaxis, internal (stomach and intestine) medicine.

Amino Acid Ad-Layers.

SMA-treated substrates can be used to create peptide, protein and otherorganic ad-layers on SMA-treated substrates. Peptide, protein and otherorganic ad-layers are useful for bio-active, bio-inert, and diagnosticsurfaces. For example, histidine has been widely used as an affinity tagto purify engineered proteins using a nickel-immobilized resin.Histidine is an amino acid containing an imidazole side chain with arelatively neutral pKa (approximately 6). The imidazole side chains andthe relatively neutral pKa mean that relatively small shifts in cellularpH will change its charge. For this reason, histidine is useful as acoordinating ligand in metalloproteins, as a catalytic site in certainenzymes, such as iron-sulfur containing oxygenase (sulfite oxygenase,rubredoxin, etc) and hemoproteins.

Histidine-tags are also often used for affinity purification ofrecombinant proteins expressed in E. coli or other prokaryoticexpression systems. Histidine-tagging is the option of choice forpurifying recombinant proteins in denaturing conditions, and can also beused to detect proteins via anti-histidine-tag antibodies in gelstaining (SDS-PAGE) with fluorescently labeled metal ions. This isuseful in subcellular localization, ELISA, Western blotting or otherimmuno-analytical methods. However, histidine-tagging cannot be used todetect protein-protein interactions under reducing conditions or incombination with EDTA and many types of detergents. The approachdescribed herein therefore represents a facile approach to linkingHis-tagged proteins onto SMA-treated substrates. This is useful forprotein immobilization because it can be a convenient way to control theorientation of immobilized proteins on surfaces, diagnostic and/orpurification purposes.

VIII. Examples

The following examples describe various new and useful embodiments ofthe present invention. While the examples refer to substrates treatedwith dopamine, it is envisioned that any SMA according to Formula I willalso be useful in the methods described herein.

General Methods and Materials.

Materials and substrate preparation. Platinum, silver, copper, andpalladium (Alfa Aesar, Ward Hill, Mass.), sapphire (Al₂O₃, Rubicon TechInc. IL), quartz (MTI crystal, MA), stainless steel, NiTi, Si (MEMCelectronics, Italy), Carbothane®, Tecoflex®, polycarbonate andpolyethylene terephthalate (PET) (McMaster Carr Inc, Chicago, Ill.),poly(styrene) (Sigma), glass (Fischer scientific), polydimethysiloxane(PDMS, Sylgard 184, Dow corning), GaAs (University Wafer, Boston,Mass.), and silicon nitride (generous donation by Dr. Keun-Ho Kim andProf H. Espinosa, Northwestern University) were cleaned ultrasonicallyin 2-propanol for ten minutes before use. Titanium (20-50 nm) and gold(20 nm deposited onto 5 nm Ti) substrates were prepared by electron beamdeposition (Edwards FL400, Boc Edwards, Sussex, UK) on Si-wafers. PDMS(Dow Corning) was prepared by mixing 10 parts of backbone and 1 part ofcuring agent and cured at 100° C. for 2 hrs.

Example 1 SMA Solution

As shown herein, simple immersion of virtually any substrate in a dilutealkaline aqueous solution of dopamine buffered to a pH typical of marineenvironments (pH >7.5) results in spontaneous deposition of a reactivemoiety on the substrate surface. In the case of dopamine, the substratesurface forms a thin adherent polymer film (FIG. 1F-1H). Atomic forcemicroscopy (AFM) indicated that the polymer film thickness was afunction of the immersion time and reached a value of up to 50 nm aftertwenty-four hours (FIG. 1G). X-ray photoelectron spectroscopy (XPS)analysis of twenty-five diverse materials coated for three hours or morerevealed the absence of signals unique to the substrate (solid bars,FIG. 1H, and FIG. 3), indicating the formation of a polymer coating of10 nm or more in thickness.

The atomic composition of the SMA-treated substrate varied little(circles, FIG. 1H), suggesting that the composition of the SMA coatingwas independent of the substrate. The nitrogen-to-carbon signal ratio(N/C) of 0.1-0.13 is similar to the theoretical value for dopamine(N/C=0.125), implying that the coating is derived from dopaminepolymerization. Gel permeation chromatography (FIG. 4) andtime-of-flight secondary-ion mass spectrometry (ToF-SIMS) (FIG. 5) alsosuggest that dopamine polymerization caused the thin adherent film toform on the substrates.

SMA was found both in solution and on the substrate, with ToF-SIMSclearly revealing signals corresponding to dihydroxyphenyl-containingpolymer fragments. Although the exact mechanism is unknown at this time,it is likely to involve oxidation of the catechol to a quinone followedby polymerization in a manner reminiscent of melanin formation, whichoccurs through polymerization of structurally similar compounds (FIG.5).

Dopamine (2 mg/mL) was dissolved in 10 mM Tris-HCl (pH 8.5), andsubstrates were dipped into the solution. pH-induced oxidation changesthe solution color to dark brown. Stirring and/or vertical sampleorientation were necessary to prevent non-specific microparticledeposition on substrates. The polydopamine-coated substrates were rinsedwith ultrapure water and dried by nitrogen gas before storage or treatedas described below for ad-layer formation. Substrates coated in thismanner remain stable on inorganic substrates unless scratched, treatedby ultrasound, or dipped in a strong acid solution (<pH 1). Coatings onsome organic substrates such as latex beads, Sephadex™ resins and somecommercial plastics remain stable even in the presence of 1 N HClcombined with ultrasound.

In another example, different conditions (pH and concentration ofdopamine) for polydopamine coating on polystyrene surfaces were used. Ata fixed concentration of 2 mg of dopamine per milliliter of 10 mM Trisbuffer, the polydopamine coating was tested as a function of pH (7.4,8.5 and 10). Also, at a fixed pH of 8.5, dopamine concentration wasvaried from 0.05 to 2 mg/ml (coating time was 15 hrs for all samples) totest the coating capability (FIG. 24).

All conditions resulted in successful polydopamine coatings except forthe coating in the 0.05 mg of dopamine per milliliter of Tris, pH 8.5.

Incubating dopamine solution at room temperature for several days (i.e.,greater than three days) prior to immersing the substrates did notproduce surface discoloration (to dark-brown) typical of polydopaminecoatings, indicating that the coating did not occur or was too thin toobserve visually. Furthermore, the modification reaction appears to beprevented under anaerobic conditions, since purging of dopamine solutionwith argon resulted in dramatically reduced solution color change andcoating formation on immersed substrates.

Analyzing polydopamine molecular weight in solution was performed on aDawn EOS (Wyatt Technology, Santa Barbara, Calif.) GPC system using amobile phase buffer (50 mM sodium phosphate, 100 mM NaCl, pH 6.5, flowrate of 0.3 mL/min) and Shodex-OH columns. The sample was filteredbefore injection (pore size 0.8 wn).

Example 2 SMA-Treated Substrates

Under oxidative conditions (e.g., pH >7.5), a dilute alkaline aqueoussolution of dopamine surprisingly modifies substrate surfaces to includereactive, adherent polydopamine nanofilms. Virtually all natural andsynthetic substrates including, without limitation, noble metals (Au,Ag, Pt and Pd), metals with native oxide substrates (Cu, stainlesssteel, NiTi shape memory alloy), oxides (TiO₂, NiTt, SiO₂, quartz,Al₂O₃, and Nb₂O₅), semiconductors (GaAs and Si₃N₄), ceramics (glass andhydroxyapatite (HAp), and synthetic polymers (polystyrene (PS),polyethylene (PE), polycarbonate (PC), polyethylene terephthalate (PET),polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS),polyetheretherketone (PEEK), and polyurethanes (Carbothane® (PU1) andTecoflex® (PU2))) (FIG. 1H) were effectively modified using theSMA-treatment of the present invention.

To date, over twenty-five substrates were successfully modified by adilute, alkaline solution comprising dopamine using an aqueous one-potprocess. Potentially any substrate known to man, including variouscomposite materials, can be used in this process. For instance, x-rayphotoelectron spectroscopy (XPS) analysis showed that substrate signalssuch as Si2p, Ti2p, and Au4d were completely suppressed by theSMA-treated substrates as described in Example 1 (Table 1). Instead,carbon, oxygen, and nitrogen signals were detected after themodification with a similar atomic composition of a nitrogen to carbonratio (experimental N/C=0.09-0.13, theoretical N/C_(dopamine)=0.125)irregardless of substrates. Unavoidable carbon contamination in ambientconditions lowered the N/C ratio in XPS.

Table 2 illustrates the substrates and corresponding atoms (bindingenergy and orbital) used as characteristic substrate peaks for XPScharacterization (as asterisk indicates synthetic, polymeric substrateswithout unique XPS signals except for carbon, nitrogen and oxygen.) Thepresence of the reactive moiety (in the case of dopamine, the reactivemoiety formed is an adherent polymeric film) on the substrates wasconfirmed by the appearance of N1s signal after SMA-treatment, as shownin FIG. 3 (399.5 eV for PS, 399.1 eV for PE, 399.7 eV for PC, 399.6 eVfor PET, and 399.8 eV for PEEK). The reactive moiety on PU-1,2 wasconfirmed by the nitrogen-to-carbon ratio after coating due to thepresence of substrate nitrogen).

TABLE 2 Substrate Binding energy (eV) (photoelectron orbital) Au84.1/84.9 (Au4f_(7/2,5/2)) Ag 369.9/373.9 (Ag3d_(5/2,3/2)) Pt 71.1/74.7(Pt4f_(7/2,5/2)) Cu 952.5/932.5 (Cu2p_(1/2,3/2)) Pd 335.1/340.5(Pd3d_(5/2,3/2)) Stainless steel 740.0/723.0 (Fe2P_(3/2,1/2)) 110₂456.5/462.4 (112p_(3/2,1/2)) NiTi 854.1/870.9 (Ni2p_(3/2,1/2)) Quartz,Glass 103 (quartz), 102(glass) (Si2p) Si0₂, Si₃N₄ 99.2/99.8(Si2p_(3/2,1/2)) Al₂0₃ 118.6 (Al_(2s)) GaAs 41.7, 106.5 (As3d_(3/2)Ga3p_(1/2)) PDMS 102.2 (Si2p) Nb205 207/209.5 (Nb3d_(5/2,3/2)) PTFE686.1 (Fis) PS* 284.7 (C1 s) PE* 284.8 (C1 s) PC* 284.7 (Cis) PET* 284.7(Cis) PEEK* 284.8 (C1 s) HAp 346.5/350.2 (Ca2P_(3/2,1/2))

Surface Characterization.

XPS spectra were obtained using an Omicron ESCALAB (Omicron,Taunusstein, Germany) with a monochromatic Al Ka (1486.8 eV) 300-W X-raysource, a flood gun to counter charging effects, and ultrahigh vacuum(−10⁻⁹ torr). The takeoff angle was fixed at 45° except as otherwisementioned. High-resolution scans were acquired to calculate the chemicalcompositions of the substrates. Time-of-flight secondary ion massspectroscopy (Physical Electronics, Eden Prairie, Minn.) was used tocharacterize the atomic composition of polydopamine coatings and metalad-layers (copper and silver). The mass spectrometer was equipped with aGa ion gun operated at 15 keV with a raster size of typically 100-200p.m. Multi-mode atomic force microscopy (Veeco Inc., Santa Barbara,Calif.) was used for imaging (tapping-mode using Si-cantilever,Veecoprobes, resonance frequency=210-240 kHz)).

Total Internal Reflection Fluorescence (TIRF) Microscopy.

Detailed experimental procedures have been described elsewhere (Qu etal. Proc. Natl. Acad. Sci. USA 101, 11298 (2004)). Briefly, an Olympus1×71 inverted fluorescence microscope (Melville, N.Y.) and a 60×objective (Olympus, N.A.=1.45 oil immersion) were used forsingle-molecule adsorption images. A 532-nm laser (New Focus 3951-20, 20mW power, San Jose, Calif.) was used as a light source. An O.D. equalsone neutral density filter was used for most experiments. The incidentlaser power was roughly 0.5 mW, illuminating a circular region of 40 1.1m in diameter. After excitation, the emitted photons were collected by afilter cube (Chroma Q560LPBS, HQ585/40M, Rockingham, Vt.), magnified bya 3.3× eyepiece and detected by a TEcooled and frame-transfer CCD(Andor, DV435-BV, South Windsor, Conn.). The protein used in thisexperiment was Cy3 conjugated Enigma homolog (Enh). The protein wasdissolved in 50 mM phosphate buffer pH 7.0 (1 1.IM) and experimentsperformed at room temperature (exposure time=33 msec).

Example 3 Untreated Substrates

In this example, substrates were tested to determine if substrates couldbe modified according to the present invention in an untreatedcondition. Accordingly, the following demonstrates that SMA-treatedsubstrates that have not been cleaned (i.e., are used as received) canbe modified to include at least one reactive moiety.

Substrates PEEK and PC were contacted with dopamine in a dilute,alkaline solution (2 mg/mL dopamine dissolved in 10 mM Tris; pH 8.5) for5 hrs. XPS was used to determine the efficacy of the SMA-treatedsubstrates. The presence of N1s signals (approximately 400 eV) (see FIG.26A-B) indicated successful polydopamine coating on unclean substrates(bare substrates do not show N1s).

These results indicate that substrates may be modified according to thepresent invention, even when such substrates are covered in paint, oil,grease, rust, protectant and the like.

Example 4 Photolithography

Dopamine's heterogeneous oxidative polymerization causes a substratetreated with dopamine to form a reactive moiety on the surface in theform of an adherent polymeric film. The polymeric film evolves inthickness as analyzed by photolithography micropatterning as a functionof time and subsequent photoresist etching. This experiment resulted insubstrates modified to include locally patterned thin films of dopamine,and the thickness was assessed by atomic force microscopy (AFM) (FIG. 6Ainset). The coating thickness increased in a time-dependent manner andevolved up to 50 nm after one day (FIG. 6A).

The chemical identity of the polydopamine coating was analyzed by timeof flight secondary ion mass spectrometry (ToF-SIMS). This techniquerelies on the ionization of chemical species (2^(nd) ions) adsorbed onsubstrates which become fragmented molecules by incident primary ionbeam (Ga⁺), and the ionized molecules are analyzed in time-of-flightdetectors. ToF-SIMS clearly proved the existence of polymerized dopamine(i.e., melanin ad-layers) by showing a fragmented trimer of5,6-dihydroxlindole and leukodopaminechrome (M 445, FIG. 6B), which aremonomeric units generated by the sequential oxidation of dopamine(reaction sequence (I)-FIG. 2A). The ToF-SIMS results also showedinteresting cleavage patterns: twice dehydroxylation (α,α;OH-phenyl-OH→phenyl-OH→phenyl) and ring opening (β; C₆H₆→C₂H₂+GPOproviding unambiguous evidence of diol and phenyl ring content in theadsorbed layers.

Photoresist (Microposit S-1818, Shipley, Marlborough, Mass.) wasspin-cast at 4000 rpm for 50 sec and then baked for 1 mM at 95° C.Utilizing a contact mask aligner (Q2000, Quintel Corp. San Jose,Calif.), the photoresist was exposed to UV (345 nm) light for sixseconds and was subsequently developed for forty sec (MF-CD-26, Shipley,Mass.). Polydopamine coating was applied to the patterned substrates forthree to six hours as described above. Finally, photoresist was removedby immersion in N-methyl-pyrrolidinone (NMP) for five to ten seconds.The coating thickness (FIG. 3) was measured by AFM on patternedsubstrates.

Example 5 SMA-Assisted Electroless Metallization

The metal binding ability of catechols present in the SMA's of thepresent invention was exploited to deposit adherent and uniform metalcoatings onto substrates by electroless metallization. Silver and coppermetal films were deposited on substrates by dip-coating SMA-treatedsubstrates into silver nitrate and copper (II) chloride solutions,respectively (FIG. 7). Metal film deposition and roughness was confirmedby XPS and ToF-SIMS analysis, which demonstrated successful metal filmdeposition on a number of ceramic, polymer and metal substratesincluding nitrocellulose, coinage metals, commercial plastics, siliconnitride, glass, gold, titanium, Si, polycarbonate, polystyrene, PEEK,gold, niobium oxide, aluminum oxide, and nickel-titanium (FIGS. 8-10).

Metal coatings were also successfully applied in this manner toSMA-treated substrates including flexible polymer substrates and bulkobjects with complex shapes (FIG. 7A-C), as well as flat substrates inwhich the SMA was patterned using standard photolithography techniques(FIG. 7D-F). Unlike many other approaches to electroless metallization,the use of (immobilized) colloidal metal seed particles was unnecessaryfor spontaneous formation of adherent metal films.

Surface-Independent Silver Deposition.

Silver has long been recognized as an anti-bacterial agent suitable formedical devices. Using the present invention, any underlying SMA-treatedsubstrate can be modified to have silver metal layers. In the case ofdopamine, silver metal layers can be formed solely by the redox power ofthe dopamine layer without a reducing agent. This implies that theunderlying polydopamine coating on the substrate oxidizes during metalion reduction. SMA-treated substrates were dipped into a 50 mM aqueoussilver nitrate solution for eighteen hours (room temperature).Substrates were then washed with ultrapure water and dried with N₂ gas.

A series of XPS spectra showed clearly differentiated signals fromsilicon nitride (FIG. 8A, top), the polydopamine coating (middle), andelectroless silver metallization afterward (bottom, reaction in 50 mMsilver nitrate in water at room temperature) indicating layer by layerdeposition at each step. Strong silver peaks (Ag_(3d) 368.9 eV red line,FIG. 8A) were detected, completely suppressing a nitrogen signal fromthe underlying dopamine layer at a take-off angle of 20° (bottom inset).

Due to the surface-independent nature of the SMA-treated substratesdescribed herein, virtually any substrate can be modified to includesilver metallization. For instance, in addition to modifying siliconnitride, silver metal was successfully deposited on severalrepresentative substrates (FIG. 8B, glass (top left), gold (top right),Ti (bottom left) and PEEK (bottom right)). This was confirmed by thecharacteristic isotopic pattern (106.9 and 108.9 m/z) of silver mass inToF SIMS.

In addition to the bulk electroless deposition, micropatterned silverdeposition was acquired by photolithography followed bypolydopamine-coating and silver metallization. The resulting silverpattern demonstrates that the metallization process described herein canbe incorporated into conventional lithography processes. Additionally,the method described above provide an aqueous, cost effective andsurface-independent preparation process that does not require toxicPd/Sn colloids for catalysis. The method presented herein thusrepresents a significant advance in electroless silver deposition.

Surface-Independent Electroless Copper Metallization.

Using the method described herein, electroless copper metal plating wasachieved on virtually all substrates, and was especially successful withsynthetic polymer substrates. For instance, polyethylene terephthalate(PET) has been used for a substrate for flexible displays, an importantcommercial substrate for future electronic devices. Integrated coppercircuit on PET substrates will supply power to organic light emittingdiodes (OLED). Contacting PET substrates with dopamine followed byelectroless copper plating is a simple and cost-effective methodpotentially revolutionizing integrated circuits.

The enediol group in dopamine has a strong affinity to various metalsincluding copper so bound copper ions on the polydopamine-coatedsubstrate act as seeds for subsequent metallization. Under a reducingcondition, metal copper was successfully deposited on varioussubstrates: Si, Al₂O₃, Nb₂O₅, NiTi, polystyrene, polycarbonate,polyetheretherketone, glass, and gold (FIG. 7). A micropatterningprocess was also developed to demonstrate a potential usage of circuitboard applications on a hard substrate (glass, FIG. 7C) and a flexiblesubstrate (cellulose acetate, FIG. 7D).

The electroless copper deposition became ineffective without the SMAtreatment, indicating the SMA-treated, substrate-bound copper plays acritical role in metallization. Substrates without SMA treatment showedstrong substrate peaks: Si2p (103.3 eV) on Si, Nb (202.4 eV) on Nb₂O₅,and F1s (685 eV) on polytetrafluoroethylene indicating partial or nometallization (FIG. 11). SMA-treated substrates were metallized throughimmersion in copper (II) or silver salt solutions.

For electroless copper plating, a solution of 50 mMethylenediaminetetraacetic acid (EDTA), 50 mM copper(II) chloride(CuCl₂), and 0.1 M boric acid (H₃BO₃) was prepared in ultrapure water,and the pH was adjusted to 7.0 using 1 N of NaOH. This solution can bestored in a refrigerator for future use. Immediately before use, 0.1 Mdimethylamine-borane (DMAB) was added to the copper plating solution,after which the SMA-treated substrates were placed in the solution fortwo to three hours at 30° C. Substrates were then washed with ultrapurewater and dried with N₂ gas.

Example 6 Biofouling and Biosensor Applications

Interfacial amino- or cysteinyl-dopamine coupling was performed bytransferring pre-SMA-treated substrates to a monofunctional PEG solution(2 mg/mL methoxy-PEG-thiol or amine 5k (mPEG-SH, mPEG-NH₂) 10 mM Tris pH8.5 50° C.). This simple two-step (SMA-treatment followed by PEGylation)aqueous chemistry successfully achieved universally-protein resistantsubstrates. The protein resistance capabilities were visualized at aresolution of a single molecule level using total internal reflectionfluorescence (TIRF) microscopy.

Unmodified glass substrates upon exposure to proteins showed massiveadsorption after 30 minutes (FIG. 12A, top), which was significantlyimproved by the traditional silane-PEG modification (bottom panel).However, long-term fouling resistance failed under the continuousexposure of a protein solution for 48 hrs (bottom right). SMA-treatedsubstrates modified in a secondary reaction to contain PEG (mPEG-NH₂, 5kDa) exhibited excellent antifouling properties showing incredibleshort- (30 min) and long-term (48 hrs) stability with virtually nodefect (middle left and right respectively).

In the focal area, only fourteen proteins were detected on theSMA-treated, PEG-modified glass substrate, whereas approximately 400proteins were adsorbed on silane-PEG modified substrates after 48 hrs.This demonstrates the enormously advantages of the present invention.

The proteins resolved by TIRF microscopy correspond to 0.01 pg/cm² whichis below the lowest limit (approximately 1 ng/cm²) of common surfaceanalytical tools such as surface plasma resonance spectroscopy oroptical waveguide light spectroscopy. Thus, PEGylated non-foulingsubstrates can be prepared in a surface-independent way. Fornon-transparent substrates, the four-hour fibroblast binding assay wasused instead of TIRF microscopy (see Example 10). Also, the assay ofcell binding resistance is an important criterion to examine antifoulingperformance of substrates in vitro.

All substrates tested, including oxide (Ti), metal (Au), semiconductor(Si₃N₄), polymer (polytetrafluoroethylene (PTFE), polyurethanes(Tecoflex®, Carbothane®)) and ceramic (glass) substrates exhibitedexcellent antifouling properties (FIG. 3B). The thiol end group of thePEG chain (mPEG-SH, 5 kDa) in this experiment was used for an elementalprobe in X-ray photoelectron spectroscopy (XPS). Sulfur 2p (163 eV)orbital signal was clearly observed (FIG. 12C), demonstrating successfulinterfacial PEG conjugations.

Example 7 SMA-Assisted SAM Formation

For alkanethiol ad-layer formation, 5 mM of dodecanethiol(Sigma-Aldrich, Milwaukee, Wis.), 1-mercapto-11-undecyl tri(ethyleneglycol) (0EG3-C11-SH), or OEG6-C11-SH (Asemblon Inc, Redmond, Wash.) wasdissolved in dichloromethane (DCM) which was pre-equilibrated bybubbling with He or N₂. SMA-treated substrates (SMA according to FormulaII) were subsequently added followed by triethylamine (finalconcentration 10 mM). After five hours or more (typically overnightreaction for eighteen hours), the SMA-treated substrates were rinsed byeither DCM or ethanol and dried with nitrogen.

An alkanethiol monolayer was spontaneously formed through simpleimmersion of the SMA-treated substrates (FIG. 13B). Monolayer formationon the polydopamine sub-layer is believed to involve reaction betweenterminal thiol groups and the catechol/quinone groups of thepolydopamine coating of the substrate in a manner analogous to thereaction between thiols and noble metal films in the formation ofconventional SAMs. Alkanethiol monolayers formed by this approach(referred to herein as “pseudo-SAMs” or “pSAMs”) appear to befunctionally similar to conventionally formed SAMs.

For example, spontaneous formation of pSAMs using methyl-terminatedalkanethiol (C12-SH) was suggested by water contact angles of greaterthan 100° (FIG. 13B, Table 3) and XPS spectra revealing the presence ofsulfur in the modified substrates (FIG. 14). Table 3 describes theevolution of contact angles of SAMs formed on variouspolydopamine-coated substrates. 0_(stat) and 0_(stat) are advancing andstatic contact angles, respectively. The average contact angles ofpolydopamine-coated and SAM-formed substrates are shown in the last row.pSAMs were formed in this way on at least seven different materialsincluding several ceramics and polymers.

TABLE 3 Bare Polydopamine SAM 0_(adv) (0_(stat)) 0_(adv) (0_(stat))0_(adv) (0_(stat)) PTFE 115 (106) 60 (49) 111 (102) PC 103 (96)  54 (42)104 (96)  NC 95 (84) 53 (41) 118 (106) SiO₂  21 (<10) 66 (54) 101 (92) TiO₂  22 (<10) 63 (51) 103 (94)  Cu 88 (78) 55 (43) 119 (109) Au 68 (54)57 (46) 101 (90)  Average — 58 (47) 108 (98) 

Example 8 PEG Grafting

In this example, at least a portion of a substrate was contacted withdopamine to form a reactive, SMA-treated substrate which was contactedwith a secondary reactive moiety to form fouling-resistant surfaces.Specifically, fouling-resistant substrates were made by covalentlygrafting amine- or thiol-terminated methoxy-poly(ethylene glycol)(mPEG-NH₂ or mPEG-SH in 10 mM Tris, pH 8.5, 50° C.) to thepolydopamine-coated substrate surface (FIG. 15).

For PEG grafting, 5 mg/mL of methoxy-poly(ethylene glycol)-thiol(mPEG-SH, 5 kDa, SunBio, Ahn-Yang, South Korea) or methoxy-poly(ethyleneglycol)-amine (mPEG-NH₂, 5 kDa, Nektar, San Carlos, Calif.) wasdissolved in 10 mM Tris pH 8.0 or sodium phosphate buffer pH 8.0. Thebuffer used for mPEG-SH was vacuum degassed for more than one hour toprevent oxidation (—S—S—) between thiol groups.

mPEG-NH₂-modified, polydopamine-coated glass substrates exhibitedsubstantial reduction in nonspecific protein adsorption compared touncoated glass, and also outperformed glass substrates modified by asilane-terminated PEG in terms of fouling resistance after two days ofcontinuous exposure to protein solution (FIG. 13D-F). Similarly, mPEG-SHgrafting onto a variety of polydopamine-coated substrates led todramatic reduction of fibroblast cell attachment compared to theunmodified substrates (FIG. 13G, Table 4). The polydopamine coatingitself was supportive of fibroblast cell adhesion at a level similar tobare substrates (for example, the total area of attached cells onpolydopamine modified SiO₂ (46±1.4×10³ μm²) was similar to unmodifiedSiO₂ (55±8.6×10³ μm²)), leading to the conclusion that the observeddecrease in cell adhesion was due to the grafted mPEG-SH.

Example 9 SMA-Treated Substrates Having Flagella Labeling

In this example, latex beads were contacted with an aqueous, alkalinesolution of dopamine as described in Example 1. The latex beads (1 μm indiameter) were spread onto pre-adsorbed E. coli, resulting in one beadattached to a flagella protein (presumably via N-terminus and lysineresidues) as evidenced by the counterclockwise rotation of the flagella(FIG. 16, box). The dots represent an individual bead non-specificallyadsorbed onto the glass surface showing no spatial movement.

Example 10 Short-Term (4 hr) Fibroblast Adhesion

NIH 3T3 fibroblasts (ATCC, Manassas, Va.) were maintained at 37° C. with5% CO₂ in Dulbecco's Modified Eagle's medium (DMEM, Cellgro, Herndon,Va.) containing 10% fetal bovine serum (FBS, ATCC, Manassas, Va.) and100 μg/ml of penicillin and 100 Wm′ of streptomycin. Trypsinized cellswere resuspended in DMEM with 10% FBS and then counted for sub-culturesand/or seeded onto the test substrates at a cell density of 5.0×10³cells/cm². After 4 hrs, cells were stained with 2.5 pM Calcein-AM(Molecular Probes) in complete PBS for one hour at 37° C. culture. Cellattachment was quantified by acquiring nine images from random locationsof each substrate using a fluorescence microscope (Olympus BX-40,2_(ex)549 nm, X_(em)=565 nm) equipped with a CCD camera (RoperScientific, Trenton, N.J.). Finally, the resulting images were processedusing Metamorph software (Universal Imaging, Downington, Pa.).

TABLE 4 # of cells Substrates # of cells (bare) (PEG-polydopamine) Glass68.7 ± 14 0 ± 0 TiO₂ 72.1 ± 13 0 ± 0 Au 62.9 ± 14 1.3 ± 1   S1₃N₄ 57.1 ±9  0 ± 0 PTFE 7.8 ± 4 0.2 ± 0.4 PU1 16.9 ± 13 0.6 ± 0.7 PU2 15.1 ± 4 0.6 ± 1.3 PS 23.6 ± 8  1.1 ± 1.6

Example 11 SMA-Assisted Grafting of Hyaluronic Acid

Ad-layers of the glycosaminoglycan hyaluronic acid (HA) were added toSMA-treated substrates prepared according to Example 1 for specificbiomolecular interactions. HA/receptor interactions are important forphysiological and pathophysiological processes including angiogenesis,hematopoietic stem cell commitment and homing, and tumor metastasis.Partially thiolated HA was grafted onto a variety of SMA-treatedsubstrates (FIG. 17) and HA ad-layer bioactivity was measured viaadhesion of the human megakaryocytic M07e cell line. Unlike fibroblasts,M07e cells did not adhere to polydopamine but did adhere to HA-graftedpolydopamine-coated substrates in a dose dependent manner (FIG. 17B).

Together with decreased binding in the presence of soluble HA (FIG.17C), these findings are consistent with expression of the HA receptorCD44 by M07e cells (FIG. 18). Polydopamine and HA-graftedpolydopamine-coated substrates were biocompatible as evidenced bysimilar levels of M07e cell expansion compared to tissue culturepolystyrene, although only the HA-grafted polydopamine-coated substratessupported cell adhesion (FIG. 170D-F; FIG. 19).

17 kDa HA (Lifecore, Chaska, Minn.) was thiolated using a previouslypublished protocol (Lee et al., Macromolecules 39, 23 (2006)). Themodified HA had approximately 50% substitution (by NMR) with thiolgroups. Thiolated HA (0.001-2 mg/mL in de-oxygenated 10 mM Tris buffer,pH 8.0) was reacted with polydopamine-coated substrates for typicallyovernight to yield HAfunctionalized substrates. HA-tethered,polydopamine-coated glass or indium-tin oxide (ITO) substrates wereattached to a bottomless sixteen-well chamber slide (Nunc, Rochester,N.Y.) via the injection of a self-curing silicone rubber (Silastic® DowCorning) gasket. For TCPS, standard ninety six-well plates were used,and the polydopamine coating and HA ad-layer formation steps wereperformed sequentially in each well. (Please note that the polydopaminecoating and HA ad-layer formation can also be performed simultaneouslyin each well.)

M07e Cell Culture.

M07e cells (DMSZ, Germany) were adapted to grow in IMDM (Sigma)supplemented with 2.5% FBS (Hyclone), 10 ng/mL GM-CSF (BerlexLaboratories), and 1 mg/mL gentamicin sulfate (Sigma). Cells weremaintained in exponential growth phase between 5×10⁵ and 1×10⁶ cells/mL.Normal-force cell adhesion assays were performed as previously described(Jensen et al., J. Am. Chem. Soc. 126, 15223 (2004)). Briefly, M07ecells were stained with 5 ti·g/mL Calcein AM in PBS and incubated innormal growth media on substrates for two hours prior to removingnon-adherent cells by inverted centrifugation at 30 rcf in sealed bagsfilled with PBS. Image analysis of pre- and post-spin images was used tocalculate the percent cell adhesion. Substrates for extended cellculture were sterilized with short-wave UV light for thirty minutesprior to seeding cells in normal growth medium at a density of 3.75×105cells/mL. Adhesion was measured on days 2 and 4 using the normal-forcecell adhesion assay. However, in this case the cells were staineddirectly in the wells via addition of 40 uL of Calcein AM (diluted to 5pg/mL PBS) thirty minutes prior to pre-centrifugation imaging. For HAcompetition, soluble 17 kDa HA was incubated with M07e cells for thirtyminutes at 37° C. prior to loading onto HA-grafted, polydopamine-coatedwells. For the M07e cell expansion assay, cell density was measured bytotal nuclei counts in a solution of hexadecyltrimethylammoniumbromide(Sigma; 30 g/L), sodium chloride (8.33 g/L) and EDTA (366.25 mg/L) witha Coulter Multisizer.

Flow Cytometry Analysis of CD44 Levels on M07e Cells.

To determine the expression levels of the HA receptor CD44, M07e cellswere washed with PBS containing 1 g/L sodium azide and 0.5% bovine serumalbumin. Allophycocyanin (APC)-conjugated mouse anti-human-CD44 antibodyor APC conjugated isotype control mouse-IgG_(2b),x antibody (BectonDickinson) were incubated with the cells for thirty minutes at roomtemperature. After washing, cells were analyzed on a Becton DickinsonLSRII flow cytometer using FACSDiva software (Becton Dickinson).

Example 12 SMA-Assisted Metal Removal

Twenty mg of dopamine hydrochloride and 1 g of beads were added to 20 mlof pH 8 10 mM Tris buffer. The solution was put on the rocker for threehours for dopamine coating. A column of SMA-treated beads was preparedaccording to Example 1 and DI water was used to remove excess dopaminehydrochloride. 4.5 ml of metal solution was added to the column. Thesolution was put on the rocker for certain time to react, and then thefiltrate was collected. The concentration of the filtrate was measuredusing ICP-AES.

Results can be seen in Table 5. Cr, Hg, and Pb showed great affinity forbinding to polydopamine-coated beads. Cu showed relatively weak binding.Cd, Ba, and Se showed no affinity for binding. The last three tests onCr, Hg, and Pb were conducted to see if the SMA-treated beads of thepresent invention could effectively remove the metal ions at lowconcentration, and the metal ion concentrations after binding can fallbelow the MCLs. When measuring such low concentrations, detection limitof the metal ion with ICP-AES and reliability of the data should beconsidered. ICP-MS can also be used to measure low concentrations.Generally, it falls within the detection limit when it shows a clearintensity peak for a certain concentration of metal ion. Further, thedata is reliable when a fit standard can be generated.

For Cr, the data show that its concentration after binding fell belowthe MCL. The intensity peak for the sample was clear. Thus,polydopamine-coated beads effectively removed Cr to below the MCL. ForPb, the data show that its concentration after binding, although veryclose, did not fall below the MCL. Further experiments were conducted totest the accuracy of this data.

Hg is the most difficult metal species for ICP-AES to accurately measurethe concentration. In ICP-AES, their intensity peaks tend to fluctuateeven for the same sample. When measuring fractions of ppm, slightchanges in intensity can lead to relatively large change in thecalculated concentration. Overall, the data presented herein illustratethat polydopamine-coated substrates can be used to remove metals such asCr, Hg and Pb from water. An increase in the amount of polymer in thepolymeric-coated substrate may likely to reduce the final concentrationto below the MCL values for each.

TABLE 5 Conc. of added Conc. Metal (reaction metal solution Conc.without Measured MCL³ time¹) (ppm) Binding² (ppm) (ppm) Cu (overnight)10 5 4.16 1.3 Pb (overnight) 10 5 2.42 0.015 Hg (2 hr) 10 5 1.1 0.002 Hg(overnight) 10 5 1.01 0.002 Cr (1 hr) 10 5 0.57 0.1 Cd (1 hr) 10 5 4.850.005 Se (1 hr) 10 5 5.22 0.05 Ba (1 hr) 10 5 5.22 2 Cr (1 hr) 1 0.50.02 0.1 Pb (1 hr) 1 0.5 0.031 0.015 Hg (1 hr) 1 0.5 0.17 0.002¹Reaction time indicates Step 4 in the procedure. ²“Concentrationwithout Binding” was obtained from a prediction that 1 g of beads holdabout 4.5 mL of water and additional 4.5 mL of metal solution shouldmake the overall concentration half the concentration of the added metalsolution. ³MCL stands for Maximum Contaminant Level for drinking waterset by U.S. Environment Protection Agency.

Example 13 SMA-Treated Substrates Surface Conjugation ofPoly-L-Histidine

Poly-histidine (0.5 mg/mL, Sigma, M_(w)=12,000) (pHis) was dissolved inacetate/phosphate/tris buffers with various pHs. Subsequently,polydopamine-coated substrates (prepared according to Example 1) wereimmersed in pHis-containing solutions buffered at various pHs (4.0 and6.8) for 4 hrs. As shown in FIG. 11A, pHis surface reaction waspH-dependent; the surface carbon composition of the polydopamine-coatedsilicon wafer reacted with pHis at low pH showed the absence of apeptide carbon (O═C—NH) signal, whereas the peptide carbon was appearedin XPS from the surface immersed in the pHis-containing neutral buffer(pH=6.8), indicating that the deprotonated nitrogen in imidazole ringsis chemically reactive toward polydopamine layers. Likewise, a newoxygen signal from peptide bonds (approximately 531 eV) was detected atthe polydopamine-coated silicon substrate reacted with pHis at pH 6.8(FIG. 20B).

The pHis-conjugated polydopamine-coated silicon surface previouslycharacterized in XPS (pH 6.8 sample) was used for time-of-flightsecondary ion mass spectrometry (ToF SIMS) analysis. As shown in FIG.21A, peaks (m/z=69, 81, 82, 95, and 110) detected from the pHisimmobilized surface are reminiscent of imidazole-containing structures,which were not detected in the polydopamine-coated surface (FIG. 21B).

Due to the difference in pKa of imidazole's secondary amine(approximately 6) and lysine's s-primary amine (approximately 10), itcan be hypothesized that the corresponding amine group from each sidechain might exhibit different reactivity onto polydopamine-coatedsubstrates depending on reaction buffer pHs. A heterobifunctionalmolecule, N-acetyl-histidine-oligo(ethylene glycol)-lysine(Ac—N-His-OEG3-Lys), was designed and synthesized by using a standardFmoc solid-phase peptide synthesis method (FIG. 22A). Matrix-assistedlaser desorption/ionization time-of-flight (MALDI TOF) mass spectrometryshowed the successful synthesis of Ac—N-His-OEG3-Lys (m/z=645.1) (FIG.22B).

The Ac—N-His-OEG3-Lys molecule (hereafter His-Lys) can be covalentlyimmobilized via either secondary amine of imidazole (polydopamine-His)or s-amine of lysine (polydopamine-Lys) side chain, which significantlyimpacts the orientation of the s-primary amine or imidazole groups withrespect to the substrate surface. The s-primary amine is exposed to anaqueous solvent if histidine reacts with a polydopamine layer, or isalternatively not exposed to the solvent as a result of lysine reactionwith the polydopamine layer, and the predominant orientation maytherefore be controlled by the pH of the medium during reaction as shownin the following example where N-hydroxysuccinimidyl biotin andsubsequent peroxidase-conjugated streptavidin coupling was used todetermine the orientation of His-Lys molecules.

Surface coupling reactions of His-Lys molecules (0.1 mM) dissolved in 10mM acetate/phosphate-/tris co-buffer, (pH 5.5, 6.4, 7.4, 8.4, and 9.5)for 5 hrs followed by biotinylation (10 mM) (4 hrs, in 10 mM phosphatebuffer pH 7.8) were performed. The colorimetric enzyme assay forperoxidase resulted in pH-dependent enzyme activities (FIG. 23A),demonstrating preferential orientation of His-Lys molecules covalentlyimmobilized on polydopamine-coated substrates (FIG. 23B-C). The enzymeactivity was monitored by the colorimetric product, purpurogallin, at420 nm in which pyrogallol and hydrogen peroxide were used assubstrates.

10 mg/mL of pyrogallol in 0.1 M phosphate buffer (pH 6.0) and a dilutehydrogen peroxide solution (1:74=H₂O₂:H₂O, v/v) were prepared assubstrates for peroxidase. The peroxidase reaction was triggered by thevertical insertion of the enzyme-immobilized polydopamine surface to aquartz cuvette. Composition of the substrate solution is as follows: 2.0mL of phosphate buffer, pH 6.0, 0.3 mL of pyrogallol solution, pH 6.0,and 0.2 mL of hydrogen peroxide solution.

Example 14 Norepinephrine-Treated Substrates

Inspired by the surface-independent coating ability of dopamine, astructural derivative of dopamine, norepinephrine, was also tested andfound to exhibit the versatile surface-modifying property. A wide rangeof substrates (noble metals, oxides, polymers, semiconductors, andceramics) were treated with norepinephrine (15-20 hrs, 2 mg ofnorepinephrine per milliliter of 10 mM tris, pH 7.5 or higher andnon-aqueous solvents such as chloroform, dichloromethane, methanol,ethanol, (iso)-propanol, dimethylformamide, dimethylsulfoxide, hexane,etc.), and subsequently the substrates were rinsed with water. Contactangle of each substrate was measured before (hatch) and after (solid)norepinephrine coating (FIG. 25).

As shown in FIG. 25, the contact angle after coating was relativelyconsistent (approximately 60°) indicating successful norepinephrinecoating, whereas the contact angles of bare materials varied fromhydrophilic (approximately 10°) to hydrophobic (approximately) 130°.

It should be noted that the above description, attached figures andtheir descriptions are intended to be illustrative and not limiting ofthis invention. Many themes and variations of this invention will besuggested to one skilled in this and, in light of the disclosure. Allsuch themes and variations are within the contemplation hereof. Forinstance, while this invention has been described in conjunction withthe various exemplary embodiments outlined above, various alternatives,modifications, variations, improvements, and/or substantial equivalents,whether known or that rare or may be presently unforeseen, may becomeapparent to those having at least ordinary skill in the art. Variouschanges may be made without departing from the spirit and scope of theinvention. Therefore, the invention is intended to embrace all known orlater-developed alternatives, modifications, variations, improvements,and/or substantial equivalents of these exemplary embodiments.

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We claim:
 1. A method of reducing amounts of metal in a fluid comprisingthe steps of: a) contacting at least a portion of a substrate with analkaline, aqueous solution under oxidative conditions, the solutioncomprising a surface-modifying agent according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and b) contacting the surface-modified substratewith a reactive moiety, wherein the reactive moiety reacts with and isbound to the modified surface c) positioning the surface-modifiedsubstrate with a reactive moiety found to the modified surface in afluid with metal, whereby the modified substrate binds to at least aportion of the metal, thereby reducing the amounts of metal in thefluid.
 2. The method of claim 1 wherein the reactive moiety is a metal.3. A method of modifying a substrate to form a biofouling-resistant,modified substrate, the method comprising the steps of: a) contacting atleast a portion of the surface of the substrate with an alkalinesolution under oxidative conditions, the solution comprising asurface-modifying agent according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and b) contacting at least a portion of thesurface-modified substrate with a biofouling-resistant reactive moiety,wherein a biofouling-resistant, surface-modified substrate is formed. 4.The method of claim 3 wherein the biofouling-resistant reactive moietyis selected from the group consisting of thiols, primary amines,secondary amines, nitriles, aldehydes, imidazoles, azides, halides,polyhexamethylene dithiocarbonate, hydrogen, hydroxyls, carboxylicacids, aldehydes, carboxylic esters or a carboxamides.
 5. The method ofclaim 3 wherein the modified substrate is part of a medical device.
 6. Akit for modifying a substrate surface, the kit comprising: a) asurface-modifying agent according to Formula I:

wherein each of R₁, R₂, R₃, R₄ and R₅ is independently selected from thegroup consisting of a thiol, a primary amine, a secondary amine, anitrile, an aldehyde, an imidazole, an azide, a halide, apolyhexamethylene dithiocarbonate, a hydrogen, a hydroxyl, a carboxylicacid, an aldehyde, a carboxylic ester or a carboxamide, provided atleast one of R₁, R₂, R₃, R₄ and R₅ is not a hydrogen atom; wherein xranges from 0 to 10 and wherein y ranges from 0 to 10, provided that xor y is at least 1; and b) instructions for use.
 7. The kit of claim 6further comprising a reactive moiety selected from the group consistingof thiols, primary amines, secondary amines, nitriles, aldehydes,imidazoles, azides, halides, polyhexamethylene dithiocarbonate,hydrogen, hydroxyls, carboxylic acids, aldehydes, carboxylic esters orcarboxamides.
 8. The kit of claim 6 further comprising a substratesurface to be modified.
 9. The kit of claim 6 wherein thesurface-modifying agent is in solution.
 10. The kit of claim 6 whereinthe surface-modifying agent is in powdered form.